Nicotianamine synthase and gene encoding the same

ABSTRACT

A nicotianamine synthase is isolated and purified. Then the gene of this enzyme is cloned and the base sequence and amino acid sequence thereof are determined. This gene is employed in constructing plants, in particular, grass plants highly tolerant to iron deficiency. A nicotianamine synthase involved in the mugineic acid biosynthesis pathway; the amino acid sequence thereof; a gene encoding the same; a vector containing this gene; cells transformed by the vector; a process for producing nicotianamine by using the same; plants transformed by the gene encoding the nicotianamine synthase; and an antibody against the nicotianamine syntase.

TECHNICAL FIELD

The present invention relates to a nicotianamine synthase involved inthe mugineic acid biosynthetic pathway, the amino acid sequence thereof,a gene encoding the same, a vector, a process for producingnicotianamine by using the same, plants transformed by the gene encodingthe nicotianamine synthase, and an antibody against the nicotianaminesynthase.

BACKGROUND ART

Graminaceous plants that absorb by chelating the insoluble state Fe(III)in soil using mugineic acid and adopt so called the Strategy-IImechanism of Fe acquisition secrete Fe chelators (phytosiderophores)from their roots to solubilize sparingly soluble Fe in the rhizosphere(Roemheld, 1987). The amount of the secreted phytosiderophores increasesunder Fe-deficiency stress. The mugineic acid family is the onlyexamples of phytosiderophores known so far (Takagi, 1976). Tolerance toFe deficiency in graminaceous plants is thought to depend on a quantityof mugineic acid family secreted by plants (Takagi et al. 1984, Roemheldand Marschner 1986, Marschner et al. 1987, Mori et al. 1987, Kawai etal. 1988, Mori et al. 1988, Mihashi and Mori 1989, and Shingh et al.1993).

The biosynthetic pathway of mugineic acid in plants is shown in FIG. 1.S-adenosylmethionine is synthesized from methionine byS-adenosylmethionine synthase. Subsequently, three molecules ofS-adenosylmethionine are combined to form one molecule of nicotianamineby nicotianamine synthase. The generated nicotianamine is then convertedto 3″-keto acid by nicotianamine aminotransferase, and 2′-deoxymugineicacid is synthesized by the subsequent action of a reductase. A furtherseries of hydroxylation steps produces the other mugineic acidderivatives including mugineic acid from the deoxymugineic acid (Moriand Nishizawa 1987, Shojima et al. 1989, Shojima et al. 1990 and Ma andNomoto 1993).

A compound in FIG. 1, a compound in the lower right, wherein R₁ and R₂are hydrogen and R₃ is hydroxyl, is mugineic acid. A compound wherein R₁is hydrogen and R₂ and R₃ are hydroxyl, is 3-hydroxymugineic acid. Alsoa compound wherein R₂ is hydrogen and R₁ and R₈ are hydroxyl, is3-epihydroxymugineic acid.

Three S-adenosylmethionine synthase genes were isolated from barleyroots, but these genes were not induced by Fe deficiency (Takizawa etal. 1996). A gene Ids3, which is obtained from the barley bydifferential screening, is suspected to be a gene, which convertsdeoxymugineic acid to mugineic acid by hydroxylation and is stronglyinduced by Fe-deficiency (Nakanishi et al. 1993). Further, nicotianamineaminotransferase was purified and isolated from Fe-deficient barleyroots, and two nicotianamine aminotransferase genes, Naat-A and Naat-B,were isolated (Takahashi et al. 1997). Naat-A expression was induced inFe-deficient roots.

The synthesis of nicotianamine from S-adenosylmethionine is similar topolyamine synthesis from decaroboxy-S-adenosylmethionine. In contrast topolyamine synthase, however, nicotianamine synthase catalyzes thecombination of three S-adenosylmethionine molecules and the azetidinering formation at the same time (FIG. 1). Such the nicotianaminesynthase is a novel type of enzyme. Previously, we reported the partialpurification of nicotianamine synthase from the roots of Fe-deficientbarley and expression pattern of the activity (Higuchi et al. 1994,Higuchi et al. 1995, Kanazawa et al. 1995, Higuchi et al. 1996a andHiguchi et al. 1996b). Since nicotianamine synthase is easily decomposedduring extraction and purification, it has been difficult to purifysufficient quantities for amino acid sequencing.

The present invention has an object to provide a plant, especiallygraminaceous plant, highly tolerant to Fe-deficiency, as a result ofisolating and purifying a nicotianamine synthase, being cloned the geneof this enzyme, determining the base sequence and amino acid sequencethereof, and using said enzyme.

DISCLOSURE OF INVENTION

The present invention relates to a nicotianamine synthase shown in SEQID NO: 1 comprising amino acid sequence shown in SEQ ID NO: 1, or aminoacid sequence having deletion in a part thereof, being substituted bythe other amino acids or being added with the other amino acids.

The present invention relates to the gene encoding said amino acidsequence of nicotianamine synthase.

The present invention also relates to a vector comprising containingsaid gene, and a transformant transformed by the said vector.

The present invention relates to a process for production ofnicotianamine using the said transformant.

The present invention further relates to plants, especially graminaceousplants, to which said gene is introduced, and fruits obtained by growingsaid plants.

The present invention relates to a process for extraction of saidnicotianamine synthase in the presence of thiol protease inhibitor,preferably E-64.

Further, the present invention relates to an antibody against saidnicotianamine synthase.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows the biosynthetic pathway of mugineic acid family.

FIG. 2 shows a comparison of nicotianamine synthase purification fromFe-dependent and control barley roots.

FIG. 3 shows a preparative SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis, hereinafter designates asSDS-PAGE) around 30–35 kDa. The horizontal bar indicates relative enzymeactivity detected from the gels.

FIG. 4 shows elution pattern of nicotianamine synthase activity from thegel-filtration column.

The large closed circles (●) indicates enzyme activity.

FIG. 5 shows a comparison with a six partial amino acid sequencedetermined by nicotianamine synthase originated from barley and similarsequence of graminaceous plants obtained by computer search of thedatabase. Identical amino acid residue is shown in “:” (SEQ ID NOS30–37, respectively in order of appearance).

FIG. 6 shows full length of HvNAS1 cDNA (SEQ ID NO: 2) and amino acidsequence (SEQ ID NO: 1) deduced therefrom. The underlined sequencesindicate the identical partial amino acid sequences of fragments in theabove FIG. 5. Numbers of the nucleotide sequence are indicated to theright of each row. Amino acid numbers are indicated on the left of eachrow.

FIG. 7 shows comparison of the deduced amino acid sequences of the above7 cDNA obtained from barley (SEQ ID NOS 7, 13, 11, 3, 5, 1, and 9,respectively in order of appearance). Asterisks “*” indicates identicalamino acid residues in all sequences used to generate consensussequences SEQ ID NOS: 23–29.

FIG. 8 shows results of thin layer chromatographic (TLC) analysis ofnicotianamine synthase activity obtained from E. coli crude extractexpressing a fused protein of maltose binding protein—HvNAS1.

FIG. 9 shows Northern—hybridization analysis of HvNAS1 as a probe.

FIG. 10 shows Southern—hybridization analysis of HvNAS1 as a probe.

FIG. 11 shows Western-blot analysis of crude enzyme used for detectionof nicotianamine synthase activity.

FIG. 12 shows Western-blot analysis of total protein extracted bytrichloroacetic acid/acetone.

FIG. 13 shows comparison of nicotianamine synthase purification fromFe-deficient barley and control barley after DEAE-Sepharose FF.

FIG. 14 shows comparison of nicotianamine synthase purification fromFe-deficient barley and control barley after Ether Toyopearl 650M.

FIG. 15 shows results of thin layer chromatographic (TLC) analysis ofnicotianamine synthase activity obtained from E. coli crude extractexpressing a fused protein of maltose binding protein—OsNAS1.

FIG. 16 shows Northern—hybridization analysis of OsNAS1 as a probe.

FIG. 17 shows results of thin layer chromatographic (TLC) analysis ofnicotianamine synthase activity obtained from E. coli crude extractexpressing a fused proteins of maltose binding protein—AtNAS1, AtNAS2 orAtNAS3.

FIG. 18 shows results of RT-PCR of total RNA extracted from theaboveground parts and roots of Arabidopsis thaliana. Right groupindicates positive control.

BEST MODE FOR CARRYING OUT THE INVENTION

We have tried to isolate nicotianamine synthase (Higuchi et al. Plant &Soil, Vol. 165, p. 173–179, 1994), and since nicotianamine synthase waseasily decomposed and was difficult to isolate and purify, we wereunable to obtain sufficient amounts of protein to determine its partialamino acid sequence. Subsequently, it was found that a thiol proteaseinhibitor E-64 (hereinafter designates as E-64) was very effective insuppressing degradation of nicotianamine synthase (Higuchi et al. Plant& Soil, Vol. 178, p. 171–177, 1996 a).

In the present invention, as a result that frozen roots were crushed toa fine powder in liquid N₂ and then rapidly homogenized with buffercontaining 0.1 mM thiol protease inhibitor E-64, nicotianamine synthaseprotein could be isolated and its gene could also be isolated.

Further, the enzyme of the present invention recovered its activity byremoval of SDS after SDS-PAGE treatment, but the rate of recovery wasvery low (Higuchi et al. Plant & Soil, Vol. 165, p. 173–179, 1994).Consequently, degree of purification should be increased up beforetreatment of SDS-PAGE. Then the column chromatography procedures werefurther improved.

We have also found that the enzyme of the present invention isrelatively hydrophobic and a buffer containing a mild surface activeagent CHAPS increased the rate of recovery. Several ion-exchangechromatography carriers were tested, and DEAE-Sepharose FF and DEAESephacel were found to be the most effective. In addition to TSK gelButyl Toyopearl, another hydrophobic chromatography carrier, TSK gelEther Toyopearl 650M, effectively removed impurities of the 30–35 kDa.

The enzyme of the present invention has been reported that it was thepeptide of 30–35 kDa, the activity of which was recovered by removingSDS after SDS-PAGE treatment, and the activity was detected as a broadmolecular weight range of 30–35 kDa (refer to FIG. 3). FIG. 3 shows aresult of preparative SDS-PAGE in the fractions showing enzyme activity.SDS-PAGE was carried out using 11% acrylamide slab gels. A portion ofthe gel was stained with Coomassie brilliant blue and the rest of thegel was stained with Cu. The gel containing proteins between 30–35 kDain size was cut into seven fragments (indicated by the short lines). Thethick bars in FIG. 3 indicate relative enzymatic activities detectedfrom each gel fragment.

In order to identify nicotianamine synthase peptide from the proteinshaving these molecular weights, the peptides, which were contained inthe nicotianamine synthase fractions, purified from Fe-deficient andcontrol barley roots were compared using SDS-PAGE. From each barley root200 g, the present enzyme was purified according to the method describedin example 3 hereinbelow.

The enzyme activity of the control was a quarter of the Fe-deficientroots.

The peptide composition of the active enzyme fraction from eachpurification step of the present enzyme was analyzed and compared bySDS-PAGE, and results are shown in FIG. 2, FIG. 13 and FIG. 14. FIG. 2,FIG. 13 and FIG. 14 show comparison with the active fraction from thepurification step of Fe-deficient barley roots 200 g [in the figure,shown with (−)], and the active fraction from the purification step ofthe control barley roots 200 g [in the figure, shown with (+)]. SDS-PAGEwas carried out using 12.5% acrylamide slab gels (Laemmli, Nature Vol.227, p. 680–685, 1970). Gels were stained with Coomassie brilliant blue.FIG. 2 shows a step before DEAE-Sepharose. The upper row shows enzymefrom Fe-deficient barley roots and the lower row shows enzyme fromcontrol roots. In each lane, lanes 1, crude extract, 200 μg of protein;lanes 2, after Butyl Toyopearl 650M, 100 μg of protein; lanes 3, afterhydroxyapatite, 20 μg of protein; and lanes 4, after Butyl Toyopearl650M, 15 μg of protein, are shown.

FIG. 13 shows after DEAE-Sepharose FF, each lane, 25 μg of protein. FIG.14 shows after Ether Toyopearl 650M; in which left shows inactivefraction, and right shows active fraction, and 1/25 of each fraction iselectrophoresed.

As a result, almost no difference was observed in both Fe-deficient andcontrol roots before DEAE-Sepharose step (refer to FIG. 2). After theDEAE-Sepharose step it became clear that the 30- and 31-kDa peptideswere induced by Fe-deficiency (refer to FIG. 13). After the EtherToyopearl step, the 31 kDa peptide was eliminated from the activefraction. The 32 and 33 kDa peptides were found to be newly induced byFe-deficiency (refer to FIG. 14). Activities were detected from the 32and 33 kDa peptides, but no activity was detected from 30 kDa peptide(refer to FIG. 3).

Molecular weight of the enzyme of the present invention was determinedby gel-filtration.

Estimated molecular weight of nicotianamine synthase by gel-filtrationwas reported to be 40,000–50,000 (Higuchi et al. Plant & Soil, Vol. 165,p. 173–179, 1994). But this did not correspond with the value estimatedby SDS-PAGE.

In the present study, the buffer containing CHAPS effectively increasedthe resolution and molecular weight of the present enzyme was estimatedto be 35,000 (refer to FIG. 4). This corresponds well to the valueestimated by SDS-PAGE.

FIG. 4 shows elution pattern of nicotianamine synthase from thegel-filtration column. The black circles (●) indicate the enzymeactivity and the solid line indicates absorption at 280 nm. The activefraction after hydroxyapatite chromatography was applied to a SephacrylS300HR (Pharmacia) column (1.5 cm×71 cm, 125 ml), equilibrated withdeveloping buffer (50 mM Tris, 1 mM EDTA, 0.1 M KCl, 0.05% CHAPS, 0.1 mMp-APMSF and 3 mM DTT, pH 8.0). Molecular weight markers used werethyroglobulin (Mr 670,000), γ-globulin (Mr 158,000), ovalbumin (Mr44,000), and myoglobin (Mr 17,000). The linear flow was 10 cm/hour.

Partial amino acid sequence was determined from purified nicotianaminesynthase.

The above explained 30 kDa, 32 kDa and 33 kDa peptides were purifiedfrom 1 kg of Fe-deficient barley roots by using a method in example 3hereinbelow. These were partially degraded using a method in example 4hereinbelow. Although 32- and 33-kDa peptides could not be completelyseparated from each other, these might have similar sequence or 32 kDapeptide was presumed to be the degradation product of 33 kDa peptide,and both of them were degraded in together.

The determined partial amino acid sequences indicated that thesepeptides were very similar in each other (FIG. 5). Further, since themolecular weights of the 33 kDa and 32 kDa (1) fragments had almostunchanged molecular weight as compared with before degradation, thissequence might be N-terminal region of the present enzyme. As a resultof computer search of the database, a gene of unknown function havingvery similar sequence to these sequences was found to exist in Oryzasativa and Alahidopsis thaliana. Especially, EST-cDNA clones D23792 andD24790 of Oryza sativa were very similar with 80.0% identity in a33-amino acid overlap in the former and 68.4% identity in a 19-aminoacid overlap in the latter (FIG. 5).

FIG. 5 shows a comparison with a six partial amino acid sequencedetermined by nicotianamine synthase originated from barley and similarsequence of graminaceous plants obtained by computer search of thedatabase. Identical amino acid residue is shown in “:”. The part ofnucleotide sequences indicated by the arrows was applied for thesequences of primer used in PCR.

Cloning and nucleotide sequences of cDNA clones encoding nicotianaminesynthase were performed and determined.

PCR amplification of total cDNA prepared from Fe-deficient barley rootsusing degenerate primers designed from the partial amino acid sequenceobtained from the method explained hereinbefore was performed, but theobjective DNA could not amplified. Then the primers having singlenucleotide sequence (shown by arrows in FIG. 5) from sequences of Oryzasativa, D23792 and D24790, were synthesized and PCR amplification wasperformed. The 205 bp fragment was amplified by PCR using NF and NRprimers and the 274 bp fragment was amplified by PCR using IF and IRprimers, and these contained the objective sequences. A cDNA libraryprepared using poly (A)⁺ RNA from Fe-deficient barley roots was screenedand 19 positive clones using the 205 bp fragment probe and 88 positiveclones using the 274 fragment bp probe were obtained.

Among the thus obtained clones, the clone designated as HvNAS1,contained a translated region of 985 bp and amino acid sequence deducedtherefrom was 328 amino acids residue, with deduced molecular weight of35,144. This corresponded well with the value estimated by SDS-PAGE andgel-filtration. The partial amino acid sequences of the 32 kDa and 33kDa peptides were included totally in HvNAS1 (FIG. 6).

FIG. 6 shows full length of HvNAS1 cDNA and amino acid sequence deducedtherefrom. The underlined sequences indicate the identical partial aminoacid sequences of fragments in the above FIG. 5. Numbers of thenucleotide sequence are indicated to the right of each row. Amino acidnumbers are indicated on the left of each row.

The predicted pI of 5.2 matched the value estimated by nativeisoelectric focusing electrophoresis well. The six clones having verysimilar sequence other than HvNAS1, i.e. HvNAS2, HvNAS3, HvNAS4, HvNAS5,HvNAS6 and HvNAS7, were also obtained (Table 1, FIG. 7).

FIG. 7 shows comparison of the deduced amino acid sequences of the above7 cDNA obtained from barley. Asterisks “*” indicates identical aminoacid residues in all sequences.

The nucleotide sequences of these clones are shown in SEQ ID NO: 2(HvNAS1), SEQ ID NO: 4 (HvNAS2), SEQ ID NO: 6 (HvNAS3), SEQ ID NO: 8(HvNAS4), SEQ ID NO: 10 (HvNAS5), SEQ ID NO: 12 (HvNAS6) and SEQ ID NO:14 (HvNAS7), respectively. The amino acid sequences of these amino acidsequences are shown in SEQ ID NO: 1 (HvNAS1), SEQ ID NO: 3 (HvNAS2), SEQID NO: 5 (HvNAS3), SEQ ID NO: 7 (HvNAS4), SEQ ID NO: 9 (HvNAS5), SEQ IDNO: 11 (HvNAS6) and SEQ ID NO: 13 (HvNAS7), respectively.

TABLE 1 Properties of nas clones Number of Identity Identity IdentityAmino Acid Molecular to nas 1 to nas 2 to nas 4 Clone Residues Weight pI(%) (%) (%) HvNAS 1 328 35144 5.20 — HvNAS 2 336 35839 5.07 72 — HvNAS 3336 36013 5.47 72 95 HvNAS 4 330 35396 4.91 73 89 — HvNAS 5 283 301485.22 61 61 59 HvNAS 6 329 35350 5.07 74 89 88 HvNAS 7 330 35244 4.98 7086 91

The partial amino acid sequences determined from the 30 kDa peptide wereall included in HvNAS5. The 5′- and 3′-non-translated regions of theseclones were not similar with each other.

D23792 and D24790 similar to nicotianamine synthase of Oryzae sativawere found with about 80% identity to HvNAS1. AC003114 and AB005245 ofArbidopsis thaliana were found with about 45% identity to HvNAS1.

The obtained HvNAS1 protein was expressed in E. coli.

The PCR amplification of HvNAS1 ORF was cloned with vector pMAL-c2 toexpress HvNAS1 fused with C-terminal of maltose biding protein. Theexpression of fused protein is strongly induced by IPTG.

The crude extract was obtained from the transformed E. coli, andnicotianamine synthase activity was assayed in the state of the fusedprotein. The crude extract from the strain transformed with only thevector could not be detected the activity, whereas in case of insertedwith HvNAS1 ORF, the activity was detected. Result is shown in FIG. 8.

FIG. 8 shows results of thin layer chromatographic (TLC) analysis ofnicotianamine synthase obtained from E. coli crude extract expressing afused protein of maltose binding protein—HvNAS1. In FIG. 8, lane 1: astandard nicotianamine synthase; lane 2: E. coli expressing maltosebinding protein (SAM); and lane 3: E. coli expressing maltose bindingprotein—HvNAS1 fused protein.

Northern hybridization analysis conducted by the method described inexample 7 hereinbelow indicated that this gene was strongly induced inFe-deficient roots (FIG. 9). This coincides with expression pattern ofthe present enzyme activity (Higuchi et al. 1994). FIG. 9 shows a resultof Northern hybridization analysis using HvNAS1 as a probe. Total RNAwas extracted from after one week of Fe-deficient treatment and controlbarley leaves and roots, and in each lane, 5 μg of RNA wereelectrophoresed.

Southern hybridization analysis of the barley genome DNA was performedaccording to the method described in example 8 hereinafter mentioned.Cutting of DNA with BamHI, EcoRI or HindIII produced plurality offragments, however none of clones obtained at present could be digestedby BamHI and EcoRI, consequently nicotianamine synthase gene might existwith multiple copies in genomes of barley and rice (FIG. 10).

FIG. 10 shows Southern—hybridization analysis of HvNAS1 as a probe.Genomic DNAs from barley and rice were digested with BamHI (lanes B),EcoRI (lanes R) and HindIII (lanes H) and 10 μg thereof wereelectrophoresed in each lane.

Further, using antigen prepared by the method described in example 9hereinbelow, Western-blot analysis was performed according to the methoddescribed in example 10. It was found that the present enzyme proteinwas rapidly decomposed during the operation in the crude extractprepared for detecting the present enzyme activity (FIG. 11). Thestaining patterns coincided with the fact that the present enzymeactivity was detected on the broad range between 30–35 kDa afterSDS-PAGE (refer to FIG. 3).

FIG. 11 shows Western-blot analysis of crude enzyme used for detectionof activity. SDS-PAGE was performed using 12.5% acrylamide slab gel.Protein 100 μg was electrophoresed.

The crude extract obtained from denatured protein according to themethod described in example 10 hereinbelow was detected as almost singleband with 35–36 kDa (FIG. 12). This value coincided with the deducedvalue from the amino acid sequence.

FIG. 12 shows Western-blot analysis of total protein extracted bytrichloroacetic acid/acetone. SDS-PAGE was performed using 12.5%acrylamide slab gel. Protein 100 μg was electrophoresed. Proteins 200 μgextracted from roots and proteins 500 μg extracted from leaves wereelectrophoresed.

Western-blot analysis after 2-dimention electrophoresis reveals todetect several spots. This coincided with the fact of obtainingplurality of nicotianamine synthase gene. All spots were induced inFe-deficient roots.

As a result that cDNA library from Fe-deficient rice roots poly (A)⁺ RNAwas screened using probes prepared by cutting HvNAS1 with restrictionenzymes ApaLI and XhoI, 20 clones were obtained. These clones weredivided into 3 types of clones according to their sequences, and amongthem, only one type contains ORF full length, which was designated asOsNAS1. Nucleotide sequence of OsNAS1 is shown in SEQ ID NO: 16 andamino acid sequence is shown in SEQ ID NO: 15.

PCR amplification of OsNAS1 ORF was cloned with a vector pMAL-c2 toexpress a form fused with maltose binding protein C-terminal. The fusedprotein is strongly induced its expression by IPTG.

Crude extract from the transformed E. coli with the fused protein wasobtained and nicotianamine synthase activity was assayed in the state ofthe fused protein. The same activity with HvNAS1 was detected. Result isshown in FIG. 15. FIG. 15 shows results of thin layer chromatographic(TLC) analysis of nicotianamine synthase obtained from E. coli crudeextract expressing a fused protein of maltose binding protein—OsNAS1. InFIG. 15, lane 1: a standard nicotianamine (NA); lane 2: an extract fromE. coli expressing maltose binding protein—OsNAS1 fused protein; andlane 3: an extract from E. coli expressing maltose bindingprotein—HvNAS1 fused protein.

Northern hybridization analysis conducted by the method described inexample 7 hereinbelow indicated that in contrast to barley, theexpression was induced in rice by Fe-deficient treatment not only inroots but also in leaves (FIG. 16). FIG. 16 shows a result of Northernhybridization analysis using OsNAS1 ORF as a probe. Total RNA wasextracted from after two weeks of Fe-deficient treatment and controlrice leaves and roots, and in each lane, 5 μg of RNA wereelectrophoresed.

Nucleotide sequence of Arabidopsis thaliana similar to HvNAS1 obtainedby computer search of the database was used as a primer. PCRamplification for genome DNA of Arabidopsis thaliana resulted to obtainthree nicotianamine synthase genes. These were designated as AtNAS1,AtNAS2 and AtNAS3.

Nucleotide sequence of these genes are shown in SEQ ID NO: 18 (AtNAS1),SEQ ID NO: 20 (AtNAS2) and SEQ ID NO: 22 (AtNAS3). These amino acidsequences are shown in SEQ ID NO: 17 (AtNAS1), SEQ ID NO: 19 (AtNAS2)and SEQ ID NO: 21 (AtNAS3).

AtNAS1, AtNAS2 and AtNAS3 ORF were amplified with PCR and were clonedwith a vector pMAL-c2. Each of them was tried to be expressed in theform of fusing with maltose binding protein C-terminal. The expressionof the fused protein was strongly induced by IPTG.

Crude extract from the transformed E. coli with the fused protein wasobtained and nicotianamine synthase activity was assayed in the state ofthe fused protein. The activity was detected. Result is shown in FIG.17. FIG. 17 shows results of TLC analysis of nicotianamine synthaseactivity obtained from E. coli crude extract expressing a fused proteinof maltose binding protein—AtNAS. In FIG. 17, lanes 1: a standardnicotianamine (NA) and S-adenosylmethionine; lanes 2: an extract from E.coli expressing only maltose binding protein; lanes 3: an extract fromE. coli expressing maltose binding protein—AtNAS1 fused protein; lanes4: an extract from expressing maltose binding protein—AtNAS2 fusedprotein; and lanes 5: an extract from E. coli expressing maltose bindingprotein—AtNAS3 fused protein.

RT-PCR was conducted according to the method described in example 11hereinbelow. It was found that AtNAS1 was expressed in the roots and theaboveground parts of Arabidopsis thaliana, whereas AtNAS2 was expressedneither in the roots nor in the aboveground parts, and AtNAS3 wasexpressed only in the roots (FIG. 18). In FIG. 18, lane M showsmolecular weight marker. Gene expression was conducted in theaboveground parts, roots and positive controls. In the figure, lanes C:AtNAS1 and AtNAS2 ORF full length were amplified; lanes 1: AtNAS1specific amplification fragments; lanes 2: AtNAS2 specific amplificationfragments; and lanes 3: AtNAS3 specific amplification fragments.

The amount of secreted mugineic acid is reported increased up to 20 mgmugineic acid/g roots dry weight/day (Takagi, 1993). Crude nicotianaminesynthase activity detected by the present inventors was sufficient tofulfill it. Since the present enzyme proteins exist in more than severaltypes and 30 kDa peptide without activity exists, it can be speculatedthat as a result of aggregation of these peptides, the constructedstructure, which is preferable for binding with 3 molecules ofS-adenosylmethionine, reveals maximum activity. The molecular weightestimated by gel-filtration was 35,000 (FIG. 4).

Increase in activity by re-aggregation of subunits has not been observedat present. Since the fused protein with maltose binding protein andsubunits showed its activity, we have at present an idea that thepresent enzyme might be a monomer. However, the possibility that largeactivity can be revealed by constructing multimer, can not completelydenied.

The reaction mechanism synthesizing nicotianamine fromS-adenosylmethionine may be similar to methyl transfer reaction usingS-adenosylmethionine as a methyl donor, and a reaction synthesizingspermidine and spermine from decarboxylated S-adenosylmethionine. Thecommon catalytic domain of these enzymes has been discussed in relationto equivalent amino acids configuration occupying similar positions inhigher-order structures (Hashimoto et al. 1998 and Schluckebier et al.1995).

In future, catalytic domain may be elucidated as the results ofcomparison with nicotianamine synthase from other plant species or X-raycrystallography.

Induction of nicotianamine synthase activity by Fe-deficiency is aspecific phenomenon in graminaceous plants, and is essential for massproduction of mugineic acid family. Oryza sativa is a plant, in whichsecretion of mugineic acid family is the least among major graminaceousplants, consequently it is very weak for Fe-deficiency in calcareoussoil.

Consequently, as a result of creating transformant Oryza sativa havingtolerance to Fe-deficiency by introducing nicotianamine synthase gene ofthe present invention into the graminaceous plants, especially Oryzasativa, and expressing large amount at the Fe-deficiency, cultivation ofrice in the calcareous soil can be possible.

Heretofore, in the graminaceous plants, nicotianamine has been thoughtto have only a role as a precursor for synthesis of mugineic acidfamily. However, since the present invention has elucidated thatnicotianamine synthase gene constituted the multiple gene family, it mayplay other important roles in the graminaceous plants.

In plants, which lack the ability to secrete mugineic acid family,except for graminaceous plants, it has been proposed that nicotianamineplays a key role as an endogenous chelator of divalent metal cations,such as Fe²⁺, Cu²⁺, Zn²⁺ and Mn²⁺, and that it contributes to thehomeostasis of those metals (Stephan et al. 1994). Consequently, it mayplay the same role in the graminaceous plants.

Nicotianamine synthase activity is not induced in dicots, and expressionof gene of the present invention may not be induced by Fe-deficiency. Wehave cloned nicotianamine synthase genes of Arabidopsis thaliana.Composition of promoter regions in these genes can elucidate themechanism of gene expression caused by Fe-deficiency, and the gene ofthe present invention may play important function not only in thegraminaceous plants but also in the dicots.

SEQ ID NO: 1 shows amino acid sequence of nicotianamine synthase of thepresent invention.

The present invention includes nicotianamine synthase having amino acidsequence shown in SEQ ID NO: 1. However, the present invention is notlimited within the above nicotianamine synthase. The nicotianaminesynthase of the present invention includes, unless it losesnicotianamine synthase activity, the peptides, in which a part of theamino acid sequence of said peptide is deleted, preferably 50% or less,more preferably 30% or less, or more further preferably 10% or less inthe total amino acids, or is substituted by other amino acids, or towhich other amino acids are further added, or in which these deletion,substitution and addition may be combined.

Nucleotide sequence coding nicotianamine synthase of the presentinvention is shown in SEQ ID NO: 2.

The present invention also includes not only a gene coding nicotianaminesynthase shown in SEQ ID NO: 2 but also genes coding nicotianaminesynthase mentioned hereinabove.

The vector of the present invention introducing the above gene is notspecifically limited, and various vectors can be introduced. Preferablevector is the expression vector.

Various cells can be transformed conventionally by using recombinantvector of the present invention. Mass production of nicotianamide can beperformed by using the thus obtained transformant. These methods arewell known in the person skilled in the art.

Examples of hosts for introducing the gene of the present invention arebacteria, yeasts and cells. Preferable host is plants, especially thegraminaceous plant.

Method for introducing gene is not limited. It can be made by usingvector or can be directly introduce in genome.

Antibody of the present invention against nicotianamine synthase can beprepared conventionally by using nicotianamine synthase of the presentinvention. Antibody can be a polyclonal antibody or, if necessary,monoclonal antibody.

Further, a selective breeding of plants, preferably graminaceous plants,can be made by using gene of the present invention. Especially, the geneof the present invention can be applied for improvement of varieties,which can grow even in Fe-deficient soil.

EXAMPLES

The following examples illustrate the present invention, but are notconstrued as limiting the present invention.

Example 1 Preparation of Plant Material

Seeds of barley (Hordeum vulgare L. cv Ehimehadakamugi No. 1) weregerminated on wet filter paper and transferred into the standardhydroponic culture solution (Mori and Nishizawa, 1987) in a glass houseat natural temperature under natural light. The pH of the hydroponicculture solution was adjusted at 5.5 by 0.5 N HCl everyday. When thethird leaves developed, the plants were transferred to the hydroponicculture solution without containing Fe. The pH was maintained at 7.0 by0.5 N NaOH everyday. The control plants were also cultured in thestandard culture solution continuously. The culture solution was renewedonce in every week. Two weeks after starting Fe-deficient treatment,when severe iron chlorosis significantly appeared on the 4th and 5thleaves, roots were harvested and frozen in liquid N₂ and stored at −80°C. until use.

Example 2 Assay of Nicotianamine Synthase Activity

Modified assay method reported previously by the present inventors(Higuchi et al. 1996a) was used. Enzyme solutions were equilibrated withreaction buffer [50 mM Tris, 1 mM EDTA, 3 mM dithiothreitol (hereinafterdesignates as DTT), 10 μM (p-amidinophenyl) methanesulfonyl fluoride(hereinafter designates as p-APMSF) and 10 μMtrans-epoxysuccinyl-leucylamido-(4-guanidino) butane (hereinafterdesignates as E-64), pH 8.7]. Buffer exchange was performed by usingultrafiltration unit, Ultrafree C3LGC NMWL10000 (Millipore Co.).S-adenosylmethionine labeled with ¹⁴C in carboxyl group (Amersham Inc.)was added to the enzyme solution at the final concentration of 20 μM andkept at 25° C. for 15 minutes. The reaction products were separated bythin layer chromatography on silica gel LK6 (Whatman Inc.) usingdeveloper (phenol:butanol:formic acid:water=12:3:2:3). Radioactivity ofthe reaction products was detected by image Analyzer BAS-2000 (Fuji FilmCo.). The protein content was assayed by Bradford method using ProteinAssay Kit (Bio Rad Inc.).

Example 3 Purification of Nicotianamine Synthase

The following operations were performed at 4° C. and E-64 was added tofractions containing nicotianamine synthase at the final concentrationof 10 μM.

The frozen roots were crushed into a fine powder in liquid N₂ andhomogenized in a household juicer with 200 ml of extraction buffer [0.2M Tris, 10 mm EDTA, 5% (v/v) glycerol, 10 mM DTT, 0.1 mM E-64, 0.1 mMp-APMSF and 5% (w/v) insoluble polyvinylpyrrolidone (PVP), pH 8.0] per100 g of roots. The homogenate was centrifuged for 30 minutes at22,500×g to obtain supernatant. Ammonium sulfate was added to thesupernatant to yield a final concentration of 0.4 M and allowed to standfor 1 hour. Again, the mixture was centrifuged for 30 minutes at22,500×g to obtain supernatant.

The supernatant was loaded onto a TSK gel Butyl Toyopearl 650M column(10 ml bed volume per 100 g of roots), equilibrated with the adsorptionbuffer [20 mM Tris, 1 mM EDTA, 3 mM DTT, 0.4 M (NH₄)₂SO₄ and 0.1 mMp-APMSF, pH 8.0] and eluted with elution buffer [10 mM Tris, 1 mM EDTA,3 mM DTT, 0.1 mM p-APMSF, 5% glycerol and 0.05% 3-[(3-chloramidopropyl)dimethyl-ammonio] propanesulfonic acid (hereinafter designates asCHAPS), pH 8.0].

KCl was added to the active fraction to give a final concentration of0.4 M, and 1 M potassium phosphate buffer (pH 8.0) was added to a finalconcentration of 1 mM of KCl. A hydroxyapatite 100–350 mesh (NacalaiTesque), equilibrated with the adsorption buffer (1 mM K-P, 10 mM KCl, 3mM DTT and 0.1 mM p-APMSF, pH 8.0), was prepared at 10 ml per protein100 mg and the fractions containing nicotianamine synthase were loaded.Nicotianamine synthase was passed through without adsorption. The passedthrough fraction was loaded onto TSK gel Butyl Toyopearl 650M column (1ml bed volume per 10 mg of protein), and nicotianamine synthase waseluted in the manner described above.

The active fraction was loaded onto a DEAE-Sepharose FF column (5 ml bedvolume per 25 mg of protein, Pharmacia) equilibrated with the adsorptionbuffer (20 mM Tris, 1 mM EDTA, 3 mM DTT, 0.1 mM p-APMSF and 0.05% CHAPS,pH 8.0) and eluted with stepwise gradient elution of potassium chlorideconcentration of 0.05 M, 0.1 M, 0.15 M and 0.2 M. Nicotianamine synthasewas eluted at 0.15 M of KCl concentration.

The active fraction was loaded onto the Ether Toyopearl 650M column (10ml bed volume per 100 g of roots), equilibrated with adsorption buffer[20 mM Tris, 1 mM EDTA, 3 mM DTT, 1.2 M (NH₄)₂SO₄ and 0.1 mM p-APMSF, pH8.0]. Nicotianamine synthase was not adsorbed and passed through fromthe column. The passed through fraction was loaded onto TSK gel ButylToyopearl 650M column and fractions containing nicotianamine synthasewas eluted. The peptides in the active fraction containing nicotianaminesynthase, which was purified by the above column chromatographictreatments, were separated by sodium dodecyl sulfate—polyacrylamide gelelectrophoresis (hereinafter designates as SDS-PAGE) using 11%acrylamide slab gels. After SDS-PAGE the gel was stained with 0.3 Mcopper chloride (Dzandu et al. 1988), and the separated bands were cutout. The gel fragments were destained with 0.25 M EDTA/0.25 M Tris (pH9.0) and homogenized with the extraction buffer (1% SDS, 25 mM Tris and192 mM glycine). Each homogenate was electroeluted with SDS-free buffer(25 mM Tris and 192 mM glycine) and peptide was recovered.

Example 4 Determination of Partial Amino Acid Sequence

The isolated nicotianamine synthase was digested chemically withcyanogen bromide (Gross 1967).

After SDS-PAGE treatment, 10-fold volume of 70% (v/v) formic acid and 1%(w/v) cyanogen bromide were added to gel fragments containingnicotianamine synthase and decomposed at 4° C. for overnight. Aftercompletion of digestion, the liquid part was collected and dried invacuo. The dried substance was dissolved in SDS-PAGE sample buffer, andallowed to stand at room temperature for overnight, then the digestedproduct was separated by SDS-PAGE using 16.5% acrylamide gel containingTricine (Schagger and Jagow, 1987). The peptides were transferred onto aPVDF membrane by electroblotting (Towbin et al. 1979) and stained withamido black. The stained bands were cut out and the amino acid sequencewas determined from N-terminal side of each peptide by Edman degradationin gas-phase sequencer (model 492A protein sequencer, Applied BiosystemsInc.).

Example 5 Cloning of Nicotianamine Synthase Genes

PCR amplification was conducted for cDNA originated from Fe-deficientbarley roots using primers, which were synthesized based on the obtainedpartial amino acid sequence. A pYH23 cDNA library prepared from the poly(A)⁺RNA of Fe-deficient barley roots was screened with the thus obtainedDNA fragments of PCR product, which was labeled with [α-³²P]dATP usingthe random primer kit (Takara Shuzo Co.), as the primers. The isolatedcDNA clones were sequenced by cycle sequencing kit (Shimadzu Bunko Co.)using Shimadzu DNA sequencer DSQ-2000L.

PCR amplification was conducted for genomic DNA of Arabidopsis thalianausing primers, which were synthesized based on nucleotide sequences ofAC003114 and AB005245 of Arabidopsis thaliana. The thus obtained DNAfragments were sequenced by cycle sequencing kit (Shimadzu Bunko Co.)using Shimadzu DNA sequencer DSQ-1000L.

The determined nucleotide sequence is shown in SEQ ID NO: 2.

Example 6 Expression of NAS1 Protein in E. coli

A fragment, in which EcoRI site was introduced into the upstream of thefirst ATG of the HvNAS1 cDNA and PstI and BamHI sites were introducedinto the downstream of the stop codon of the HvNAS1 cDNA, was amplifiedby PCR. The thus obtained amplified product was subcloned in thepBluescriptII SK− using EcoRI site and BamHI site, and the correctnucleotide sequence was confirmed. The fragment between EcoRI site andPstI site was cloned into pMAL-c2 to make expression in the form offusing the HvNAS1 to the C-terminal of maltose binding protein.

A fragment, in which EcoRI site was introduced into the upstream of thefirst ATG of the OsNAS1 and HindIII site was introduced into thedownstream of the stop codon of the OsNAS1, was amplified by PCR. Thethus obtained amplified product was subcloned in the pBluescriptII SK−using EcoRI site and HindIII site, and the correct nucleotide sequencewas confirmed. The fragment between EcoRI site and HindIII site wascloned into pMAL-c2 to make expression in the form of fusing the OsNAS1to the C-terminal of maltose binding protein.

A fragment, in which EcoRI site was introduced into the upstream of thefirst ATG of the AtNAS1, AtNAS2 and AtNAS3 and XbaI site was introducedinto the downstream of the stop codon of the AtNAS1, AtNAS2 and AtNAS3,was amplified by PCR. The thus obtained amplified products weresubcloned in the pBluescriptII SK−, and the correct nucleotide sequenceswere confirmed. The fragment between EcoRI site and XbaI site was clonedinto pMAL-c2 to make expression in the form of fusing the AtNAS1, AtNAS2and AtNAS3 to the C-terminal of maltose binding proteins, respectively.

E. coli strain XL1-Blue was used as a host for expressing the said fusedprotein. pMAL-c2-HvNAS1 and pMAL-c2, respectively, were introduced intoYL1-Blue. The thus obtained recombinant bacteria were cultured in LBmedium containing ampicillin and tetracycline, each 50 μg/ml, at 37° C.until the OD 600 of the culture reached 0.5. Isopropylβ-D-thiogalactopyranaoside (IPTG) was added to the final concentrationof 0.3 mM, and continuously cultured at 37° C. for 3 hours, andcollected bacterial cells. Cells were suspended in 10 mM Tris buffercontaining 0.2 M NaCl, 1 mM EDTA, 3 mM DTT and 0.1 mM E-64, pH 7.4 andfrozen with liquid nitrogen. This was melted in ice water andultrasonication for 15 seconds was repeated for 10 times. Nicotianaminesynthase activity of the thus obtained crude extract was assayedaccording to the method described in example 2 and the enzyme activitywas confirmed.

Example 7 Northern Hybridization

Northern hybridization of barley RNA was performed using DNA fragment,which was prepared by cutting HvNAS1 cDNA with HindIII and NotI andlabeled with [α-³²P]dATP, as a probe. Total RNA was extracted frombarley (Naito et al. 1988). The extracted RNA was separated by 1.4%agarose gel electrophoresis, and blotted onto Hybond-N⁺ membranes(Amersham). Northern hybridization of rice RNA was performed usingOsNAS1 ORF, which was labeled with [α-³²P]dATP, as a probe. Total RNAwas extracted from rice. The extracted RNA was separated by 1.4% agarosegel electrophoresis, and blotted onto Hybond-N⁺ membranes (Amersham).The membrane was hybridized with the probe in 0.5 M Church phosphatebuffer (Church and Gilbert 1984), 1 mM EDTA, 7% (w/v) SDS with 100 μg/mlsalmon sperm DNA at 65° C. for overnight. The membrane was washed withbuffer containing 40 mM Church phosphate buffer and 1% (w/v) SDS at 65°C. for 10 minutes. After the washing was repeated once again, themembrane was washed with buffer containing 0.2×SSPE and 0.1% (w/v) SDSat 65° C. for 10 minutes. Radioactivity was detected using the imageanalyzer BAS-2000.

Results are shown in FIG. 9 and FIG. 16.

Example 8 Southern Hybridization

Genomic DNA was extracted from leaves of barley and rice. The extractwas digested with BamHI, EcoRI or HindIII, separated on a 0.8% (w/v)agarose gel electrophoresis, and transferred onto Hybond-N⁺ membranes(Amersham). The hybridization was performed according to the methoddescribed in example 7 and radioactivity was detected.

Result is shown in FIG. 10.

Example 9 Preparation of Polyclonal Antibody

Two rats were immunized using the antigen containing about 100 μg ofisolated nicotianamine synthase. The antigen was the same sample as thatdetermined the partial amino acid sequence. The complete Freund'sadjuvant was used at the first immunization and the incomplete Freund'sadjuvant was used since the second immunization. All the constituents ofthe blood were corrected after the rats were immunized four times, andthe obtained serum was preserved at −80° C.

Example 10 Western Blotting Analysis

Total protein was extracted using trichloroacetic acid and acetone(Damerval et al. 1986). The plants were crashed in the liquid nitrogenuntil powder was obtained, and mixed with acetone containing 0.1% (v/v)2-mercaptoethanol. The protein was precipitated by allowing to stand at−20° C. for 1 hour, and the precipitate was collected by centrifugationat 16,000×g for 30 minutes. The precipitate was suspended in acetonecontaining 0.1% (v/v) 2-mercaptoethanol and allowed to stand at −20° C.for 1 hour, then collected the precipitate by centrifugation at 16,000×gfor 30 minutes. The precipitate was dried in vacuo, and dissolved in thesample buffer [9.5 M urea, 2% (w/v) Triton X-100 and 5% (v/v) 2-ME],then centrifuged at 16,000×g for 10 minutes to obtain the supernatant.The proteins contained in the supernatant were separated by SDS-PAGE orthe denaturing two-dimensional electrophoresis (O'Farrell 1975) andtransferred onto PVDF membrane. Western blotting analysis was performedby applying the primary antibody containing anti-nicotianamine synthaseantibody prepared in example 9 and the secondary antibody containinghorse radish binding anti-mouse IgG (H+L) goat antibody (Wako PureChemicals Co.) on the membrane and coloring with diaminobenzidin.

Result is shown in FIG. 12. SDS-PAGE was performed using 12.5%acrylamide slab gel. Protein 100 μg was electrophoresed. Proteins ofroots 200 μg and leaves 500 μg were electrophoresed.

Example 11 RT-PCR

Total RNA was extracted from Arabidopsis thaliana. RT-PCR was performedwith 1 μg RNA as a template by using the EZ rTth RNA PCR kit (ParkinElmer Inc.). Specific primers for AtNAS1, AtNAS2 and AtNAS3,respectively, were used.

Result is shown in FIG. 18.

INDUSTRIAL APPLICABILITY

Various cells are transformed according to the conventional method byusing recombinant vectors of the present invention. Mass production ofnicotianamine can be performed by using the obtained transformant. Thesemethods can be performed according to the method known in the personskilled in the art.

Selective breeding of plants, preferably graminaceous plants can also beperformed using genes of the present invention. Especially, genes of thepresent invention can be applied for improving varieties, which can growon Fe-deficient soil.

1. An isolated or purified enzyme exhibiting nicotianamine synthaseactivity, wherein the enzyme comprises a polypeptide that is at least90% identical to SEQ ID NO:1.
 2. The isolated or purified enzyme ofclaim 1, wherein the enzyme comprises the polypeptide having the aminoacid sequence of SEQ ID NO:
 1. 3. The enzyme according to claim 1,wherein the enzyme comprises the consensus amino acid sequence of₁₉₉DVVFLAALVGM₂₀₉ (SEQ ID NO: 27).
 4. The enzyme of claim 1, wherein thepolypeptide further comprises all of the conserved amino acid residuesof SEQ ID NO: 1 that is: L(11), K(14), I(15), 1(22), L(25), L(28),P(30), L(37), F(38), L(41), V(42), C(45), P(47), D(52), V(53), Q(61),M(63), R(64), L(67), I(68), C(71), A(74), E(75), L(78), E(79), H(81),L(86), D(90), P(92), L(93), H(95), L(96), F(99), P(100), Y(101), N(104),Y(105), L(108), L(111), E(112), L(115), L(116), A(129), F(130), G(132),S(133), G(134), P(135), L(136), P(137), S(140), L(143), A(144), H(147),L(148), F(153), N(155), A(162), N(163), A(166), L(169), R(180), M(181),F(183), T(185), L(195), D(199), V(200), V(201), F(202), L(203), A(204),A(205), V(207), G(208), M(209), K(214), H(220), L(221), H(224), M(225),G(228), A(229), L(231), R(239), F(241), L(242), Y(243), P(244), V(246),G(255), F(256), V(258), L(259), V(261), H(263), P(264), V(268), N(270),S(271), K(277) (SEQ ID NO:29).
 5. The enzyme of claim 1, wherein thepolypeptide has more than 95% identity with the amino acid sequence ofSEQ ID NO:
 1. 6. The enzyme of claim 1, wherein the nicotianaminesynthase activity is measured in an assay in a comparison with theenzyme of SEQ ID NO:1.
 7. The enzyme of claim 1, wherein the enzyme isisolated or purified from barley.
 8. A mutated enzyme exhibitingnicotianamine synthase activity, wherein the enzyme: a. is a polypeptidehaving more than 95% identity with the amino acid sequence of SEQ ID NO:1, comprising at least one consensus sequence of SEQ ID NO: 1 that is:(1)₂₅LPXLSPSPXVDRLFTXLVXACVPXSPVDVTKL₅₆ (SEQ ID NO: 23)(2)₆₇LIRLCSXAEGXLEAHY₈₂ (SEQ ID NO: 24) (3)₉₂PLDHLGXFPY₁₀₁ (SEQ ID NO:25) (4)₁₂₈VAFXGSGPLPFSS₁₄₀ (SEQ ID NO: 26) (5)₁₉₉DVVFLAALVGM₂₀₉ (SEQ IDNO: 27) (6)₂₅₃RGGFXVLAVXHP₂₆₄ (SEQ ID NO: 28); and b. has more than 25%of the relative nicotianamine synthase activity of the enzyme of SEQ IDNO:
 1. 9. The enzyme of claim 8, wherein the polypeptide furthercomprises all of the conserved amino acid residues of SEQ ID NO: 1 thatis: L(11), K(14), I(15), I(22), L(25), L(28), P(30), L(37), F(38),L(41), V(42), C(45), P(47), D(52), V(53), Q(61), M(63), R(64), L(67),I(68), C(71), A(74), E(75), L(78), E(79), H(81), L(86), D(90), P(92),L(93), H(95), L(96), F(99), P(100), Y(101), N(104), Y(105), L(108),L(111), E(112), L(115), L(116), A(129), F(130), G(132), S(133), G(134),P(135), L(136), P(137), S(140), L(143), A(144), H(147), L(148), F(153),N(155), A(162), N(163), A(166), L(169), R(180), M(181), F(183), T(185),L(195), D(199), V(200), V(201), F(202), L(203), A(204), A(205), V(207),G(208), M(209), K(214), H(220), L(221), H(224), M(225), G(228), A(229),L(231), R(239), F(241), L(242), Y(243), P(244), V(246), G(255), F(256),V(258), L(259), V(261), H(263), P(264), V(268), N(270), S(271), K(277)(SEQ ID NO: 29).
 10. The enzyme of claim 8, wherein the nicotianaminesynthase activity is measured in an assay in a comparison with theenzyme of SEQ ID NO:1.
 11. The enzyme of claim 8, wherein thepolypeptide has more than 97% identity with the amino acid sequence ofSEQ ID NO:
 1. 12. The enzyme of claim 1, wherein the polypeptide hasmore than 97% identity with the amino acid sequence of SEQ ID NO:
 1. 13.An isolated, purified, or mutated enzyme exhibiting nicotianaminesynthase activity, wherein the enzyme comprises an active fragment ofthe amino acid sequence of SEQ ID NO: 1, the active fragment comprisinga polypeptide, wherein the polypeptide: a. comprises at least oneconsensus sequence of SEQ ID NO: 1 that is: (1)₂₅LPXLSPSPXVDRLFTXLVXACVPXSPVDVTKL₅₆ (SEQ ID NO: 23) (2)₆₇LIRLCSXAEGXLEAHY₈₂ (SEQ ID NO: 24) (3) ₉₂PLDHLGXFPY₁₀₁ (SEQ ID NO: 25)(4) ₁₂₈VAFXGSGPLPFSS₁₄₀ (SEQ ID NO: 26) (5) ₁₉₉DVVFLAALVGM₂₀₉ (SEQ IDNO: 27) (6) ₂₅₃RGGFXVLAVXHP₂₆₄ (SEQ ID NO: 28); and b. has more than 25%of the relative nicotianamine synthase activity of the enzyme of SEQ IDNO:1.
 14. An isolated or purified barley enzyme exhibiting nicotianaminesynthase activity, wherein: a. the enzyme is: i. isolated or purifiedfrom barley; or ii. expressed directly or indirectly from a nucleic acidisolated or purified from barley; or iii. expressed directly orindirectly from a chimeric nucleic acid at least partially isolated orpurified from barley; b. the enzyme comprises a polypeptide having atleast 90% identity with the amino acid sequence of SEQ ID NO: 1,comprising at least one consensus sequence of SEQ ID NO: 1 that is: (1)₂₅LPXLSPSPXVDRLFTXLVXACVPXSPVDVTKL₅₆ (SEQ ID NO: 23) (2)₆₇LIRLCSXAEGXLEAHY₈₂ (SEQ ID NO: 24) (3) ₉₂PLDHLGXFPY₁₀₁ (SEQ ID NO: 25)(4) ₁₂₈VAFXGSGPLPFSS₁₄₀ (SEQ ID NO: 26) (5) ₁₉₉DVVFLAALVGM₂₀₉ (SEQ IDNO: 27) (6) ₂₅₃RGGFXVLAVXHP₂64 (SEQ ID NO: 28); and c. the enzyme hasmore than 25% of the relative nicotianamine synthase activity of theenzyme of SEQ ID NO:
 1. 15. The enzyme of claim 14, wherein thepolypeptide further comprises all of the conserved amino acid residuesof SEQ ID NO: 1 that is: L(11), K(14), I(15), I(22), L(25), L(28),P(30), L(37), F(38), L(41), V(42), C(45), P(47), D(52), V(53), Q(61),M(63), R(64), L(67), I(68), C(71), A(74), E(75), L(78), E(79), H(81),L(86), D(90), P(92), L(93), H(95), L(96), F(99), P(100), Y(101), N(104),Y(105), L(108), L(111), E(112), L(115), L(116), A(129), F(130), G(132),S(133), G(134), P(135), L(136), P(137), S(140), L(143), A(144), H(147),L(148), F(153), N(155), A(162), N(163), A(166), L(169), R(180), M(181),F(183), T(185), L(195), D(199), V(200), V(201), F(202), L(203), A(204),A(205), V(207), G(208), M(209), K(214), H(220), L(221), H(224), M(225),G(228), A(229), L(231), R(239), F(241), L(242), Y(243), P(244), V(246),G(255), F(256), V(258), L(259), V(261), H(263), P(264), V(268), N(270),S(271), K(277) (SEQ ID NO: 29).
 16. The enzyme of claim 14, wherein thepolypeptide has more than 90% identity with the amino acid sequence ofSEQ ID NO:
 1. 17. The enzyme of claim 14, wherein the polypeptide hasmore than 95% identity with the amino acid sequence of SEQ ID NO:
 1. 18.The enzyme of claim 14, wherein the nicotianamine synthase activity ismeasured in an assay in a comparison with the enzyme of SEQ ID NO:1. 19.An isolated or purified enzyme exhibiting nicotianamine synthaseactivity, wherein the enzyme consists of the polypeptide set forth asSEQ ID NO:
 1. 20. The isolated or purified enzyme of claim 1, comprisingat least one consensus sequence of SEQ ID NO: 1 selected from the groupconsisting of: (1) ₂₅LPXLSPSPXVDRLFTXLVXACVPXSPVDVTKL₅₆ (SEQ ID NO: 23)(2) ₆₇LIRLCSXAEGXLEAHY₈₂ (SEQ ID NO: 24) (3) ₉₂PLDHLGXFPY₁₀₁ (SEQ ID NO:25) (4) ₁₂₈VAFXGSGPLPFSS₁₄₀ (SEQ ID NO: 26) (5) ₁₉₉DVVFLAALVGM₂₀₉ (SEQID NO: 27) (6) ₂₅₃RGGFXVLAVXHP₂₆₄ (SEQ ID NO: 28); and b. has more than25% of the relative nicotianamine synthase activity of the enzyme of SEQID NO:1.